The holy grail of life is written in four letters in one's genome. The ability to read and understand the 4-lettered text holds the key to understanding of life in general and all its quirky details. Two methods emerged in the 1970s to decipher the exact sequence of the four nucleotides; Guanine (G), Adenine (A), Thymidine (T) and Cytosine (C) in a sequence of DNA. The chemical sequencing method, developed by Gilbert and Maxam, is based on chemical modification of DNA and subsequent cleavage at specific sites (Maxam A M, Proc. Natl. Acad. Sci. U.S.A. 74 (2): 560-4 (1977)). The chain termination method, developed by Sanger, makes use of dideoxynucleotide triphosphates (Sanger F, Proc Natl Acad Sci USA. 74(12): 5463-5467 (1977)). Owing to its relative ease and reliability, Sanger sequencing has become the method of choice.
The Sanger reaction requires a single stranded DNA template, a primer, a DNA polymerase, four normal deoxynucleotide triphosphates (dNTPs) to extend the primer, and four dideoxynucleotide triphosphates (ddNTPs) to terminate DNA strand elongation, resulting in DNA fragments of varying length. In the classic Sanger reaction, ddNTPs are radiolabeled and final DNA fragments are separated by electrophoresis in polyacrylamide gel and visualized by autoradiography, allowing direct reading of the DNA sequence from the autoradiograph. Tagging the primer, and particularly tagging the ddNTPs, with fluorescent dye set the stage for automated DNA sequencing (Smith L M, et al., Nucl. Acids Res. 13(7): 2399-2412 (1985); Smith L M, et al., Nature 321:674-9 (1986)).
Modifications to basic Sanger sequencing include methods and products for combined amplification and sequencing reactions, such as the BigDye series by Applied Biosystems Inc. (ABI). The protocol for use of BigDye and similar amplification and sequencing techniques is as follows. Double stranded DNAs are denatured by heat to single stranded templates, annealed to a proper primer, and Taq DNA polymerase then extends the template with dNTPs and terminates elongation with ddNTPs. The ddNTPs are base-labeled with energy-transfer fluorescent dyes, which can be excited at one wave length and emits light of different wavelength. By repeated thermal denaturing and DNA synthesis, a pool of DNA fragments, each ending with a single fluorescently labeled ddNTP, is generated. The fragments are separated by capillary electrophoresis, visualized under laser as electronic chromatograms. The DNA sequence can then be read automatically using a number of software packages.
Sanger sequencing enabled sequencing of the first human genome. However, many challenges remain to be tackled, such as template-related sequencing difficulties. For example, DNA secondary structure, repetitive sequences, long stretches of homopolymers, circular DNA, and tightly-adhered sequences represent some of the many sequence-related obstacles faced in Sanger sequencing methods. There is a great need to overcome these and other template-related obstacles to improve sequencing methods.